Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an important cause of hemorrhagic colitis and the hemolytic uremic syndrome (HUS). EHEC colonizes the large intestine and secretes a potent cytotoxin called Shiga toxin (Stx) that disrupts protein synthesis eventually leading to kidney damage characteristic of HUS. Infection begins by ingestion of food or water that is contaminated with EHEC, often from contact with cattle feces, the primary reservoir of the organism. The extremely infectious dose required for disease, ca. 10 - 100 viable organisms, complicates control of transmission. Previous work in the principal investigator's laboratory resulted in the identification of the crucial intestinal colonization factor called intimin, the discovery of a large pathogenicity island encoding a type III secretion system that mediates the characteristic attaching and effacing intestinal histopathology, the characterization of a master regulator of virulence genes in this pathogen called Ler, and the discovery that key virulence factors in this pathogen are regulated by quorum sensing. The proposed research goals for the next period of support include continued characterization of the pathogenic mechanisms of EHEC as well as studies aimed at identifying key factors that are involved in maintenance of this pathogen in the bovine reservoir. The specific aims are 1) Characterize the Ler regulon and assess in vivo expression of Ler-regulated genes in a simulated human intestinal environment and bovine intestinal tract using microarrays and quantitative real time PCR;2) Characterize human host epithelial cell transcriptional responses to infection with wild type EHEC and isogenic effector mutants using in vitro organ culture (IVOC);3) Characterize the role of urease in pathogenesis and environmental persistence;and 4) Characterize transcriptional of the LEE4 operon, which encodes the the type III secretion system translocon. The proposed studies should yield significant new information regarding the pathogenesis of disease, particularly the initial steps in establishing intestinal colonization, as well as provide insights into the mechanisms responsible for maintenance of EHEC in the bovine reservoir.